Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript would be much improved by including neutralization experiments and Pseudovirion challenge in mice demonstrating protective efficacy in an established in vivo model.

Futher, the authors should define to what extent the enhanced baculovirus expression system increases the yield over current systems.

Minor points:

Materials: indicate use of spinner flasks and suspension culture. Was pluronic used in culture medium?

Line 131: specify individual truncations of all L1 ORFs.

Was residual DNA content in the VLP preps examined?

lines 172 to 185: delete detailed description of SDS-PAGE and Western blot

line 206: is Aluminum hydroxide meant by 'alum'? Which dose per injection was used?

line 224: specify ELISA plates. specify buffer, time and temp for VLP coating

line 133: were ELISA done with commercial or in-house VLP ELISA? be consistent with text in results

Figure 1: is hard to read, increase size. Figure 1 legend: delete 1x 'construction'.

Figure 2 legend: M, Protein? unclear. MW-Marker?

line 319 should read: ....which were NOT observed in the Gardasil-9...

Figure 5B:

• is very hard to read due to small size.
• line 305 states Adjuvant alone, fig 5 states PBS as control?
• why is 'HPV6' stated as immunogen for all types? please clarify

line 391: define PBNA

line 404: bur-den

 

Author Response

Comments 1: The manuscript would be much improved by including neutralization experiments and Pseudovirion challenge in mice demonstrating protective efficacy in an established in vivo model.

Response 1: We sincerely thank the reviewer for the valuable suggestion to include neutralization assays and pseudovirion challenge experiments to further confirm the protective efficacy of the vaccine candidates.

At this stage, establishing the pseudovirion production system and conducting in vivo challenge experiments require additional time and dedicated technical personnel, which are currently limited in our laboratory. To overcome this limitation, we have initiated collaboration with an external research group specializing in HPV pseudovirion models. These experiments are planned for our subsequent study to extend and strengthen the findings presented in the current work.

We fully agree that such analyses will provide important supporting evidence, and we plan to include them in future publications.

 

Comments 2: Further, the authors should define to what extent the enhanced baculovirus expression system increases the yield over current systems.

Response 2: We thank the reviewer for this important comment. While a comparison with previously reported expression yields was briefly mentioned in the original manuscript, we agree that this description was not sufficiently quantitative or explicit.

In the revised manuscript, we have now clarified the comparison by providing a broader and more balanced overview of reported yields in baculovirus expression systems. Conventional BEVS platforms typically yield HPV L1 proteins in the range of approximately 10 mg/L, while some optimized systems have reported yields up to 40 mg/L depending on the serotype and production conditions.

In contrast, the enhanced BEVS employed in this study achieved yields ranging from approximately 20 to 90 mg/L across the twelve HPV L1 serotypes. This corresponds to an approximate 2- to 5-fold increase in productivity compared to conventional systems.

We have also clarified that this improvement is likely attributable to the combined effects of the p6.9 promoter, hr3 enhancer, and burst sequences incorporated into the expression vector, which are known to enhance transcriptional activity and protein expression levels.

These points have now been incorporated into the revised Discussion section to more clearly highlight the productivity advantage and practical significance of our platform.

 

Comments 3: Materials: indicate use of spinner flasks and suspension culture. Was pluronic used in culture medium?

Response 3: Agree. In the present study, insect cells were cultured in suspension using Erlenmeyer shaking flasks (1 L flasks) under agitation conditions, rather than spinner flasks. No Pluronic F-68 was supplemented during cell culture. These details have now been clarified in the Materials and Methods section to improve reproducibility.

 

Comments 4: Line 131: specify individual truncations of all L1 ORFs.

Response 4: Agree. The truncation sites of each HPV L1 construct have now been clearly defined in the revised Supplementary Data. Specifically, we have added a table summarizing the amino acid ranges, truncation types, and the number of residues removed at the N- and C-termini for each construct to provide a clearer overview of the modifications.

 

Comments 5: Was residual DNA content in the VLP preps examined?

Response 5: We thank the reviewer for raising this important point. In this study, we attempted to measure residual host cell and viral DNA after purification; however, no meaningful levels could be detected. Although we did not employ highly sensitive methods such as qPCR at this stage, spectrophotometric analysis using a NanoDrop instrument did not detect measurable DNA concentrations in the purified samples.

Based on these findings, and given the chromatography-based purification steps implemented in our process, we consider that host cell DNA and related impurities are likely reduced to negligible levels. A more accurate and quantitative assessment of residual DNA using sensitive analytical methods will be conducted in future clinical development studies to support process validation and regulatory requirements.

 

Comments 6: lines 172 to 185: delete detailed description of SDS-PAGE and Western blot

Response 6: Agree. The descriptions of SDS-PAGE and Western blot analysis have been shortened in the revised manuscript by removing detailed procedural steps and retaining only the essential information, in order to improve clarity and conciseness.

Comments 7: line 206: is Aluminum hydroxide meant by 'alum'? Which dose per injection was used?

Response 7: We used the Alhydrogel® adjuvant 2% product and handled it according to the manufacturer’s instructions. This information has been added to and clarified in the Materials and Methods section.

 

Comments 8: line 224: specify ELISA plates. specify buffer, time and temp for VLP coating

Response 8: We used a commercially available ELISA kit, and the detailed information (manufacturer and catalog number) has been included in the Materials and Methods section of the revised manuscript.

 

Comments 9: line 133: were ELISA done with commercial or in-house VLP ELISA? be consistent with text in results

Response 9: We appreciate the reviewer’s insightful comment. In our preliminary experiments, an in‑house VLP‑based ELISA was used for initial assay optimization; however, all ELISA data presented in the current manuscript were obtained using a commercial HPV VLP ELISA kit to ensure objectivity and reproducibility of the results. Accordingly, the Methods and Results sections have been revised to clearly reflect the use of the commercial kit and to maintain consistency throughout the manuscript.

 

Comments 10: Figure 1: is hard to read, increase size. Figure 1 legend: delete 1x 'construction'.

Response 10: Agree. Figure 1 has been revised to improve readability by increasing the overall size and adjusting the layout. In addition, the figure legend has been corrected by removing the duplicated wording and revising panel (A) to “Schematic representation of the HPV L1 transfer vector.” These changes improve the clarity and presentation of the figure.

 

Comments 11: Figure 2 legend: M, Protein? unclear. MW-Marker?

Response 11: The figure legend has been corrected to clarify that “M” refers to the molecular weight marker. This change improves clarity for readers.

 

Comments 12: line 319 should read: ....which were NOT observed in the Gardasil-9...

 

Response 12: Agree. The sentence has been revised to clearly convey the intended meaning by specifying that serotype-specific IgG responses against the additional types were not observed in the GARDASIL®9 or OPT‑9 groups.

 

Comments 13: Figure 5B: is very hard to read due to small size.

Response 13: Agree. Figure 5 has been revised to improve readability by increasing the overall size and adjusting the layout and font size.

 

Comments 14: line 305 states Adjuvant alone, fig 5 states PBS as control?

Response 14: Agree. The negative control has been clarified in the Materials and Methods section as alum alone, and the corresponding description in the figure has been aligned accordingly.

 

Comments 15: why is 'HPV6' stated as immunogen for all types? please clarify

Response 15: Agree. The incorrect labeling of “HPV6” across all panels has been corrected to reflect the appropriate serotypes.

 

Comments 16: line 391: define PBNA

Response 16: Agree. In addition, the abbreviation “PBNA” has now been defined in the manuscript

 

Comments 17: line 404: bur-den

Response 17: Agree. the typographical error “bur-den” has been corrected to “burden.”

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents the development of a 12‑valent HPV L1 VLP vaccine candidate produced using an enhanced baculovirus expression vector system (BEVS). The authors provide a comprehensive workflow—from vector construction and antigen expression to physicochemical characterization and murine immunogenicity. The study is technically solid, well organized, and addresses an important global health need, particularly the challenge of HPV vaccine accessibility in low‑ and middle‑income countries. The enhanced BEVS platform and the expanded antigenic coverage beyond the licensed 9‑valent vaccine represent meaningful contributions.

Overall, the manuscript is promising and of potential interest to the field. I offer the following comments to help strengthen clarity, rigor, and presentation.

1. In Figure 1B, the PCR products for all twelve serotypes appear as very consistent bands between 1400 and 1600 bp. Since Table 2 indicates that the theoretical molecular weights of the L1 proteins vary slightly (from 51.9 to 56.4 kDa), it would be very helpful to include the specific expected base pair (bp) lengths for each gene in the figure legend to provide more precise experimental evidence.
2. The manuscript notes that variations in Western blot band intensity may reflect differences in the cross-reactivity of the CamVir-1 antibody (Figure 2B). To strengthen this inference, please clarify in the Results section whether a loading control or standardized protein quantification was used to ensure that these differences are not due to minor variations in protein loading.
3. In the DLS size distribution plots, the X-axis (Size d.nm) scales currently show slight visual variations in their starting points. Standardizing the horizontal axis range and the vertical scale across all twelve panels (e.g., from 0.1 to 1000 nm) would allow readers to more intuitively compare the monodispersity and particle size among different serotypes (Figure 3).
4. In Figure 4, certain panels (such as HPV31 and 33) exhibit lower contrast, making it harder to distinguish the VLPs from the background. It is suggested to adjust the contrast/brightness for better visibility and to explicitly state the length of the scale bar (e.g., 200 nm) in the legend rather than only noting its color.
5. Table 2 shows a significant range in yield, from 22 mg/L for HPV58 to 90 mg/L for HPV31. While this is attributed to solubility, adding a brief sentence in the Results section regarding the recovery rate during purification or the specific performance of the enhanced promoter for high-yield types would further emphasize the commercial potential of your platform.
6. In the ELISA results (Figure 5B), some statistical significance markers (e.g., asterisks) overlap with error bars or top brackets, which affect legibility. Adjusting the position of these markers slightly upward and briefly explaining the biological significance of the low background observed in OPT-9/12 for non-shared serotypes would highlight the specificity of the induced antibodies.
7. The Discussion could be strengthened by briefly commenting on the observed stability of the reassembled VLPs (e.g., qualitative stability at 4°C). Even a short note would help readers assess their suitability for downstream formulation development.
8. Since multivalent HPV vaccines may exhibit antigenic interference, it would be valuable to discuss:

whether any serotype showed reduced assembly or recovery in the mixed formulations,

whether DLS or TEM was performed before and after antigen mixing, and

if not, whether this is planned for future studies.

This will help readers evaluate the robustness of the 12‑valent formulation.

9. The study relies on ELISA IgG titers, which are informative but not fully predictive of protective efficacy. Please consider adding a short explanation regarding:

why did ELISA choose this stage,

whether pseudovirus‑based neutralization assays (PBNA) are planned, and

the known correlation between ELISA and neutralization titers in HPV vaccine research.

This will help contextualize the immunogenicity findings.

This manuscript presents a well‑executed study with clear relevance to HPV vaccine development and global vaccine accessibility. The data is promising, and the enhanced BEVS platform appears to be a strong candidate for scalable, cost‑effective production. Addressing the points above will further improve clarity, strengthen the scientific narrative, and enhance the manuscript’s overall impact.

Author Response

Comments 1: In Figure 1B, the PCR products for all twelve serotypes appear as very consistent bands between 1400 and 1600 bp. Since Table 2 indicates that the theoretical molecular weights of the L1 proteins vary slightly (from 51.9 to 56.4 kDa), it would be very helpful to include the specific expected base pair (bp) lengths for each gene in the figure legend to provide more precise experimental evidence.

Response 1: We thank the reviewer for this helpful comment. In the revised manuscript, the expected PCR product sizes (bp) for each HPV L1 gene have been indicated below each lane in Figure 1B to provide clearer experimental validation.

In addition, detailed information on primer sequences and corresponding amplicon sizes has been provided in the Supplementary Data and referenced in the Materials and Methods section.

 

Comments 2: The manuscript notes that variations in Western blot band intensity may reflect differences in the cross-reactivity of the CamVir-1 antibody (Figure 2B). To strengthen this inference, please clarify in the Results section whether a loading control or standardized protein quantification was used to ensure that these differences are not due to minor variations in protein loading.

Response 2: We thank the reviewer for this important comment. To ensure consistent comparison across samples, equal amounts of purified HPV L1 proteins (4 μg per lane) were used for analysis. This information has now been added to the Materials and Methods section.

 

Comments 3: In the DLS size distribution plots, the X-axis (Size d.nm) scales currently show slight visual variations in their starting points. Standardizing the horizontal axis range and the vertical scale across all twelve panels (e.g., from 0.1 to 1000 nm) would allow readers to more intuitively compare the monodispersity and particle size among different serotypes (Figure 3).

Response 3: We thank the reviewer for this helpful comment. The DLS plots were generated using consistent axis ranges; however, we acknowledge that differences in panel layout may have given the impression of inconsistent scaling.

To address this, the figure has been revised by adjusting the margins and layout to ensure that the axis scales are aligned consistently across all panels, thereby improving visual clarity and comparability.

 

Comments 4: In Figure 4, certain panels (such as HPV31 and 33) exhibit lower contrast, making it harder to distinguish the VLPs from the background. It is suggested to adjust the contrast/brightness for better visibility and to explicitly state the length of the scale bar (e.g., 200 nm) in the legend rather than only noting its color.

Response 4: We thank the reviewer for this helpful comment and agree that the image quality in certain panels could be improved.

In the revised manuscript, all TEM images have been enlarged to enhance visibility. In addition, for HPV31 and HPV59, alternative images from the same samples have been selected to provide clearer visualization of the VLP structures. For HPV35, the image contrast has been adjusted to improve clarity.

These revisions improve the overall quality and interpretability of Figure 4.

 

Comments 5: Table 2 shows a significant range in yield, from 22 mg/L for HPV58 to 90 mg/L for HPV31. While this is attributed to solubility, adding a brief sentence in the Results section regarding the recovery rate during purification or the specific performance of the enhanced promoter for high-yield types would further emphasize the commercial potential of your platform.

Response 5: We thank the reviewer for this insightful comment. In the revised manuscript, we have further clarified the interpretation of production yield differences among HPV serotypes in the Discussion section.

Specifically, we have expanded the discussion to note that the observed variation in yields may be influenced by intrinsic properties of each L1 protein, such as solubility, folding efficiency, and VLP assembly behavior, which can affect recovery during downstream purification.

In addition, we have emphasized that the enhanced expression system, incorporating the p6.9 promoter, hr3 enhancer, and burst sequences, contributes to the overall high productivity observed across multiple serotypes.

 

Comments 6: In the ELISA results (Figure 5B), some statistical significance markers (e.g., asterisks) overlap with error bars or top brackets, which affect legibility. Adjusting the position of these markers slightly upward and briefly explaining the biological significance of the low background observed in OPT-9/12 for non-shared serotypes would highlight the specificity of the induced antibodies.

Response 6: We thank the reviewer for this helpful comment. In the revised manuscript, the size and resolution of Figure 5B have been adjusted and the positions of the statistical significance markers have been slightly shifted to improve legibility. In addition, we have added a brief statement in the Results section noting that the low background reactivity against non‑shared serotypes in the OPT‑9/12 groups suggests minimal cross‑reactivity and indicates a high degree of serotype-specific protection potential.

 

Comments 7: The Discussion could be strengthened by briefly commenting on the observed stability of the reassembled VLPs (e.g., qualitative stability at 4°C). Even a short note would help readers assess their suitability for downstream formulation development.

Response 7: We thank the reviewer for this helpful suggestion. A brief description of the stability of the reassembled HPV L1 VLPs has been added to the Discussion section. Specifically, we observed that the VLPs retained their structural integrity when stored at 4 °C over the course of the experiments, indicating good short‑term stability. We have briefly mentioned this point in the Discussion and fully agree that a more comprehensive evaluation of long‑term stability under various storage conditions is important, which will be addressed in future studies.

 

Comments 8: Since multivalent HPV vaccines may exhibit antigenic interference, it would be valuable to discuss:

whether any serotype showed reduced assembly or recovery in the mixed formulations, whether DLS or TEM was performed before and after antigen mixing, and if not, whether this is planned for future studies. This will help readers evaluate the robustness of the 12 valent formulation.

Response 8: We appreciate the reviewer’s insightful comment regarding potential antigenic interference in multivalent HPV vaccines. Based on our current data, we observed indications of antigenic interference when comparing the mixed 12‑valent formulation with the corresponding monovalent antigens, and this point has now been briefly discussed in the revised Discussion section. We also note that a more detailed evaluation of antigen interference, including additional immunization studies specifically designed to dissect potential interactions among serotypes, will be necessary and is planned for future work.

In addition, physicochemical characterization of the mixed formulations, such as DLS or TEM analysis before and after antigen mixing, was not performed in the present study. We agree that such analyses are important to evaluate the structural stability and robustness of the multivalent formulation. These studies are planned for future work to further optimize and validate the 12-valent vaccine formulation.

 

Comments 9: The study relies on ELISA IgG titers, which are informative but not fully predictive of protective efficacy. Please consider adding a short explanation regarding:

why did ELISA choose this stage, whether pseudovirus‑based neutralization assays (PBNA) are planned, and the known correlation between ELISA and neutralization titers in HPV vaccine research.

This will help contextualize the immunogenicity findings.

Response 9: We thank the reviewer for this insightful comment. ELISA was selected at this stage as a primary screening tool to evaluate antigen-specific humoral immune responses across multiple serotypes in a high-throughput and comparative manner. This approach is commonly used in early-stage vaccine studies to assess overall immunogenicity prior to more functionally complex assays.

We agree that ELISA IgG titers do not fully reflect protective efficacy, and therefore pseudovirion-based neutralization assays (PBNA) are planned as a next step to evaluate the neutralizing activity of the induced antibodies for each HPV serotype.

Importantly, previous studies on HPV vaccines have demonstrated a positive correlation between ELISA-measured binding antibody titers and neutralizing antibody responses, although the strength of this correlation may vary depending on the serotype and assay conditions. Thus, ELISA results in this study provide a useful preliminary indication of immunogenicity, which will be further validated by PBNA in future work.

Reviewer 3 Report

Comments and Suggestions for Authors

In this study the authors address an important topic yA 12-valent HPV L1 virus-like particle vaccine candidate produced using an enhanced baculovirus expression vector system: process development, physicochemical characterization, and immunogenicity in mice. Despite this some points should be addressed before publishing.

Title it is long title please shorten and define acronyms.

Abstract please define unknown acronyms and add some numerical values of the results.

Introduction please add more details on cervical cancer global and regional epidemiology, risk factors, malignant transformation, prevention and therapeutic modilities. Moreover, add disadvantages of classical HPV vaccines. As well, add advantages of VLP. Vaccines. At the end of the introduction section please add the study rationale and aims in a clear statements.

Methods, please add the details characterization of VLP vaccines in the term of particle size, zeta potential and PDI.

Please add the details of purification of the prepared VLP and percent of yield products.

In immunization, please add allergies, hypersensitivity and mortality rate of mice if present.

Results, please include the statiscal data in the figures and tables legends in the term of data expression significant difference samples number and p value.

Discussion please refine this section and support your work with similar finding and add the contradictory if present. Moreover discuss  the similarities and differences between the nice and human immunization. Moreover discuss the advantages of VLP vaccines over classical HPV vaccines. Please discuss the study limitations and novelty.

Conclusions expand to cover all the study results.

Please check the manuscript for minor grammar errors and syntax.

Please check the manuscript for misuse of acronyms.

Please check the manuscript for the long sentences or paragraphs without references citation.

Please check the references list and update up to 2026 citation dated.

Comments on the Quality of English Language

Please check the manuscript for minor grammar errors and syntax.

Author Response

Comments 1: Title it is long title please shorten and define acronyms.

Response 1: We appreciate the reviewer’s valuable suggestion regarding the title length. In line with this comment, we have simplified the title to improve clarity and conciseness. The revised title now reads: “Development of a 12-valent HPV L1 virus-like particle vaccine using an enhanced baculovirus expression system.”

 

Comments 2: Abstract please define unknown acronyms and add some numerical values of the results.

Response 2: We thank the reviewer for this helpful suggestion. In the revised Abstract, undefined acronyms have been clarified at first mention. In addition, rather than presenting detailed numerical values in the Abstract, we now emphasize the key qualitative findings, while the exact quantitative data are provided in the main text and tables for clarity and completeness

 

Comments 3: Introduction please add more details on cervical cancer global and regional epidemiology, risk factors, malignant transformation, prevention and therapeutic modilities. Moreover, add disadvantages of classical HPV vaccines. As well, add advantages of VLP. Vaccines. At the end of the introduction section please add the study rationale and aims in a clear statements.

Response 3: We thank the reviewer for this helpful suggestion. In the revised manuscript, we have added a brief statement in the Introduction section to emphasize both the strengths of currently licensed HPV vaccines and their limitations in type coverage.

 

Comments 4: Methods, please add the details characterization of VLP vaccines in the term of particle size, zeta potential and PDI.

Response 4: We thank the reviewer for this helpful comment. In the revised Methods section, we have clarified that the particle size of the HPV VLPs was characterized by dynamic light scattering, and the corresponding polydispersity index (PDI) values were determined to assess the degree of size homogeneity of the preparations. Zeta potential was not measured in the present study, but we agree that surface charge characterization would provide additional insight into colloidal stability and intend to address this aspect in future formulation studies .

 

Comments 5: Please add the details of purification of the prepared VLP and percent of yield products.

Response 5: Agree. The purification procedure has been described in detail in the Materials and Methods section, and the yield of each VLP has been accurately reported in Table 2.

 

Comments 6: In immunization, please add allergies, hypersensitivity and mortality rate of mice if present.

Response 6: Agree. No adverse effects, including allergy, hypersensitivity, or mortality, were observed during the study period. This information has now been added to the revised manuscript.

 

Comments 7: Results, please include the statiscal data in the figures and tables legends in the term of data expression significant difference samples number and p value.

Response 7: Agree. In the revised manuscript, we have applied this recommendation by updating all relevant figure and table legends to specify the data presentation format, sample size, statistical tests used, and the corresponding p-value thresholds for significance.

 

Comments 8: Discussion please refine this section and support your work with similar finding and add the contradictory if present. Moreover discuss  the similarities and differences between the nice and human immunization. Moreover discuss the advantages of VLP vaccines over classical HPV vaccines. Please discuss the study limitations and novelty.

Response 8: Agree. In the revised manuscript, the Discussion section has been substantially expanded and refined. We now relate our findings more explicitly to previously published studies, briefly address areas where results differ, discuss similarities and differences between murine and human HPV immunization, and highlight the advantages of VLP-based vaccines over classical HPV vaccines. In addition, we have added a concise description of the main limitations of our work and clearly articulated the novelty and potential impact of this 12-valent VLP vaccine platform.

 

Comments 9: Conclusions expand to cover all the study results.

Response 9: Agree. In the revised manuscript, the Conclusions section has been expanded to more comprehensively reflect all major findings of the study.

Comments 9: Please check the manuscript for minor grammar errors and syntax.

Response 9: Agree. The manuscript has been carefully re‑checked for minor grammatical and syntactic errors, and we have revised the text accordingly. In addition, the entire manuscript has undergone professional English editing to further improve clarity and readability.

 

Comments 10: Please check the manuscript for misuse of acronyms.

Response 10: Agree. The instances where abbreviations were not properly defined or indicated in the manuscript have been corrected in the revised version.

 

Comments 11: The instances where abbreviations were not properly defined or indicated in the manuscript have been corrected in the revised version.

Response 11: Agree. We have carefully checked the manuscript for long sentences or paragraphs that lacked appropriate reference citations and have revised them accordingly. Where necessary, we have added citations or restructured the text to ensure that all substantive statements and data are properly supported by references.

 

Comments 12: Please check the references list and update up to 2026 citation dated.

Response 12: We thank the reviewer for this thoughtful suggestion regarding the inclusion of more recent references. We carefully reviewed the current literature up to 2026 and considered potential additions. However, we concluded that no additional 2026 references were essential to support the main scientific conclusions or to improve the coherence of the narrative in the present manuscript. For this reason, we did not incorporate new references beyond those already cited, which we believe adequately reflect the current state of knowledge relevant to our study. Nonetheless, we fully agree on the importance of continuously updating the literature base, and we will carefully consider newly emerging studies in this area when preparing future work.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

- lines 18-19 Background/Objectives: Cervical cancer, predominantly driven by persistent infection  with high-risk human papillomaviruses (HPVs), is most common malignancies among women worldwide.

This is not good English and is not correct: Cx is NOT most common femal cancer

line 171: remove dot at beginning of line

 

Author Response

Comments 1: - lines 18-19 Background/Objectives: Cervical cancer, predominantly driven by persistent infection with high-risk human papillomaviruses (HPVs), is most common malignancies among women worldwide.

This is not good English and is not correct: Cx is NOT most common femal cancer

Response 1: We respectfully thank the reviewer for this valuable comment. In response, we have revised the sentence as follows:

“Cervical cancer, predominantly driven by persistent infection with high‑risk human papillomaviruses (HPVs), is one of the most common malignancies and an important cause of cancer‑related mortality among women worldwide.”

 

Comment 2: line 171: remove dot at beginning of line

Response 2: We appreciate the reviewer’s comment. We have removed the dot at the beginning of line 171 as suggested.